This application proposes the continuation of a molecular genetic analysis of the mobile pathogenicity islands, SaPIs, encoding SEB, TSST-1, and other superantigens and pathogenicity factors. Our laboratory has discovered and characterized many of these elements in S. aureus, and we have recently demonstrated natural SaPI transfer to Listeria monocytogenes, as well as to other staphylococcal species. This transfer may generate Listeria derivatives with enhanced virulence. The SaPIs are discrete 15-20 kb DNA segments that occupy specific chromosomal sites. They are induced to excise and replicate by certain staphylococcal phages and are efficiently packaged into infectious particles for transmission. Specific Aims are 1. To elucidate the internal regulatory circuitry of the SaPI genome Under this aim, we will analyze SaPI gene expression and replication dynamics during the ERP cycle. It is hypothesized that following induction, SaPI genes are expressed in an explicit temporal sequence that varies according to induction scenario. We propose to test this hypothesis by analyzing the temporal pattern of SaPI gene expression (transcription pattern) under 4 different paradigms: SaPI induction by superinfecting or SOS-induced phage; incoming SaPI along with or in the absence of helper phage. 2. To analyze the SaPI-phage interface. The SaPI interacts with its inducing phage in two basic ways: it uses just those phage products that are necessary to enable the formation of infective SaPI particles, and it interferes with phage development ensuring that there is a very low probability that any potential recipient cell will be infected by an active phage particle as well as by a SaPI particle. Firstly, the SaPI uses one or more phage functions to inactivate its repressor, resulting in excision and replication; it remodels the phage capsid proteins to form its specific small particles; it diverts the phage packaging system to promote packaging of SaPI genomes at the expense of phage genomes. Secondly, the SaPI directly interferes with phage maturation and it ensures that the phage DNA is packaged mostly into small SaPI capsids, resulting in defective phage particles. Under this aim, we address both parts of this SaPI strategy. 3. To investigate the role of SaPIs in the microbiosphere. Under this aim, we will analyze the behavior of SaPI DNA during its replication cycle in S. aureus, evaluate the consequences of regulatory mutations, and analyze the phenomenon of displacement of a resident by an incoming SaPI. We will also study the interactions between co-resident SaPIs. Also under this aim, we will investigate SaPI biology in L. monocytogenes and other organisms to which it may be transferred, and we will determine the prevalence of SaPIs among different bacterial species in addition to clinical S. aureus isolates. PUBLIC HEALTH RELEVANCE: Staphylococci are extremely difficult to control owing to their remarkable ability to acquire and transmit genes for antibiotic resistance and for virulence. Our project is focused on genetic units that carry toxin genes and are transferred at very high frequencies, even to other species. By understanding the biology of these gene transfer systems, it is our hope to be able to control staphylococcal gene transfer and so reduce the danger of staphylococcal disease. [unreadable] [unreadable] [unreadable]